Device

Part:BBa_K4170055:Design

Designed by: Alexandros Giannopoulos Dimitriou   Group: iGEM22_Thessaloniki_Meta   (2022-09-30)


SUMO-LbuCas13a coding device under rhaB promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 289
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1318
    Illegal BglII site found at 5543
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1586
    Illegal AgeI site found at 2406
    Illegal AgeI site found at 3621
  • 1000
    COMPATIBLE WITH RFC[1000]


Cloning strategy

Step 1

  • PCR amplification with Ep and Sp standard primers from Basic SevaBrick Assembly [seva 3.1] using the rhamnose Repos (BBa_K4170054) as a template. This PCR produces the rhaR-rhaS part ready for Golden Gate assembly.
  • PCR amplification with Cas13a RBS FWD and P4 RVS primers using the SUMO-LbuCas13a Repos (BBa_K4170014) as a template. This PCR produces the Cas13a-RBS-P4 RVS part ready for Golden Gate assembly.
  • PCR amplification with Ev and Pv standard primers from Basic SevaBrick Assembly [seva 3.1] using the Bba_J364007 part of the 2022 DNA distribution Kit. This PCR produced the pSB1C3 backbone linearized and ready for Golden Gate assembly.

    • Step 2

    Golden Gate assembly of the PCR amplified rhaR-rhaS and Cas13a-RBS-R4 RVS parts with the linearized pSB1C3 vector for the efficient construction of the SUMO-LbuCas13a coding sequence under the transcriptional control of the rhamnose promoter.


Source

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References